Transcript 1st = 3 x 7kT. Practically 100% Efficient 2nd : Free energy in ATP

Protein sequence—from primary to quaternary
Protein Folding
https://artofbiochemistry.wordpress.com/2013/02/20/1226
/
https://www.studyblue.com/notes/note/n/campbel
l-biology-ch-5-the-structure--function-of-largebiological-molecules/deck/11460700
How does a Protein go from unfolded to folded
a) at all; b) in 1 msec; c) with no (help), chaperones?
(Helping proteins)

Unfolded
Inactive

Folded
Active
Hans Frauenfelder,
founder of biological
physics.
Main driving forces:
1) Shield hydrophobic (black spheres) residues/a.a. from water;
2) Formation of intramolecular hydrogen bonds.
Active areas: 4 centuries on it
Predicting tertiary structures from primary sequence still not solved!
Difficulty relating to experimental observations.
The Protein Energy Landscape
Largely from Martin Gruebele, Chemistry, Physics UIUC
Also from Maria Spies, Biochemistry, UIUC
Levinthal’s Paradox
Protein folding – the process that results in acquisition of the native structure from a
completely or partially unfolded state
Protein folding cannot proceed by purely random search
among ALL possible conformations:
Imagine:
100 aa protein (M.W. 10kDaltons– very small)
Let’s say 3 configurations for each step
How Many possible configurations?  3100
It takes at minimum 10-15 sec for each step:
(time scale required for bond rotation)
How long to fold?
 longer than the age of the universe!!!
Protein Folding Summary
• Proteins are made as a string of amino acids,
supposedly unstructured, and then fold up into it’s
shape.
• Can fold and do say fairly fast (< second).
• In most cases, don’t need help. In complicated cases
(big proteins, very crowded conditions such as in a
cell) proteins get help: proteins called chaperones.
• ΔG is almost always small: (5-10 kT—few H-bonds).
E goes down; S goes down. They compensate.
• Kinetics – fast cause not huge barriers. (Detailed
calculations necessary.)
• Protein Funnel is a good model.
Proteins: A short, hard life.
A typical protein folding equilibrium
constant Keq ≈ 3600.

Aunfolded
kf
 Afolded
kuf
2 weeks
(typical)
Keq= [Afolded]/[A] unfolded
= kf / kuf
This means a protein is
unfolded for how much
time/day?
24 times/day
= once/hr!
Hydrophobic regions
become exposed, becomes
ubiquinated. Reused aa in
proteasomes.
≈1 hr (if Keq=3600)
Not nearly enough chaperones to
help re-fold. Tend to do this by itself.
20-60% are natively unfolded– bind
to negatively charged substrate and
then folds.
50-100 aa
Let’s say you have protein Keq = 1000
So what fraction of states are folded?
So what’s ∆G? Keq = exp(-∆G/kT)
∆G =7 kBT
How many hydrogen bonds is this?
Free energy
Simple Calculation of ∆G from Keq.
-1
0
x
1
That’s equivalent to just a couple of Hydrogen bonds.
∆G is (almost flat).
How can this be? What about ∆E, ∆S? (Recall: ∆G = ∆E – T∆S)
If ∆S is large and ∆E is large, then ∆G can be small.
This is what happens: ∆E, T∆S ≈ -100’s kJ/mole ~ 33 kBT/molecule
(Lots of bonds form but loss of a lot of entropy,  ∆G can be small)
Protein folding: the energy landscape theory
Unfolded state
ENTROPY
Is this “free energy”, or
“energy = enthalphy”?
Intermediate states
ENERGY
IA
Molten
Globule
State
Ans: Energy (Enthalpy)
IB
Native state
Protein folding: the energy landscape theory
1. Fast – (on a ms timescales for single
domains). Unfolded proteins “roll
downhill” towards smaller populations
of conformations.
2. Highly cooperative – intermediates are
rarely observed
3. Heterogeneity of the starting points –
each unfolded molecule has different
conformation and different path downhill
the folding funnel
4. In many cases is coupled to translation
Example: the lattice model
A simplified model of protein folding:
Only 2-D motion allowed; only 90˚ motion.
(Real proteins are 3D; are not restricted to 90˚ rotation.)
• 6-mer peptide (2 hydrophobic and 4 hydrophilic amino acids)
• Each amino acid is represented as a bead
– Black bead: hydrophobic (H)
– White bead: hydrophilic (P)
• Bonds represented by straight lines
• H-H (=1 KJ/mole = 1/3 kBT/molecule) and P-P (=250J) bonds favorable
• Single 90˚ rotation per time step allowed.
What about length-dependence:
do peptides (short proteins) fold-up?
Note: Proteins fold; Peptides don’t fold
Peptides have too few H-H and P-P to fold stably.
Based on work from Ken A. Dill, 1989, and Peter Wolynes, 1987
Core and surface
solvent
solvent
solvent
solvent
solvent
solvent
solvent
solvent
solvent
solvent
solvent
(shown: a configuration
with favorable E = <H>)
Chirality in Amino acids
Although most amino acids can exist in
both left and right handed forms, Life on
Earth is made of left handed amino
acids, almost exclusively. Why?
Not really known. Meteorites have
http://en.wikipedia.org/wiki/File:C
left-handed aa.
hirality_with_hands.jpg
Alpha helix is a right-handed coil, with left-handed
amino acids. (There is steric hinderance for a lefthanded helix from left-handed amino acids.)
Similar for b-sheets.
• In 2D: To avoid issues with chirality, all molecules are made so
that the first two amino acids go upwards.
• Also, the first kink always goes to the right.
Rotation rules under Lattice Model
• 2-D model - no rotations allowed.
[Don’t allow over-counting: horizontal
= vertical configuration]
• Molecules are only allowed to
change in a single “kink” in 90˚
increments per time.
Note: these two
states would be
equivalent by an
out-of-plane
rotation, but this
is not allowed.
The Journey
Conformation Analysis
[Add up E, S = kb lnW]
E
Energy = 0 kJ
W=14
S=Rln(14)≈22JK-1mol-1
0
Energy = -0.25 kJ; -0.5 kJ
W=7
-0.5 kJ
S=Rln(7)≈16JK-1mol-1
Energy = -1 kJ
W=2
S=Rln(2)≈5.8 JK-1mol-1
Reaction
Coordinate
x
0.33
Kinetic trap
(Have to break
two bonds)
Energy = -1.5 kJ
W=1
S=Rln(1)=0
Note: Only nearest neighbors that count
Molecular Dynamics has actually taken over to make it more realistic
0.66
1
The Protein Folding funnel
Entropy
E
k ln14
k ln1 = 0
Entropy : horizonal scale
Entropy vs. Energy
(correlated monotonic function)
Ln 14
Entropy
The folded state (-1.5kJ) has the lowest
entropy, and the unfolded states have the
highest entropy
Ln 1
-1500
-1000
Energy (kJ)
-500
0
Entropy
Entropy vs. Reaction Coordinate
0
0.33
0.66
Reaction Coordinate
1.0
0.99
Free Energy Analysis (200°K)
0
Free Energy (G)
Downhill folding (but in reality, at 200K,
nothing moves)
At low temperatures, the lowest free energy
state is the most ordered state, in this case the
native state.
0
0.33
Reaction Coordinate
x
0.66
1.0
Free Energy Analysis (298°K)
Free Energy (G)
At room temperature, the folded state (-1500J)
has the lowest free energy, and thus is the
most energetically favorable conformation to
be formed.
Downhill folder
0
0.33
0.66
Reaction Coordinate
1.0
0.99
Free Energy Analysis (2000°K)
At very high temperatures, the fully denatured
state has the lowest free energy.
Free Energy
Downhill unfolder
0
0.33
0.66
Reaction Coordinate
1.0
0.99
Free Energy Analysis (360 °K)
Free Energy (G)
This is likely the equilibrium of 50:50 where
they are interconverting and equally stable.
Two state folder
Unfolded state—has some
structure
0
0.33
0.66
Reaction Coordinate
1.0
0.99
Summary of Protein Folding
Proteins can fold.
Don’t need chaperones.
ΔG is always about zero.
Kinetics – fast cause not huge barriers
Class evaluation
1. What was the most interesting thing you learned in class today?
2. What are you confused about?
3. Related to today’s subject, what would you like to know more about?
4. Any helpful comments.
Answer, and turn in at the end of class.